Project Summary/Abstract This proposal describes a four year career development plan and a research strategy for Dr. Robert Orchard to transition from a postdoctoral fellow to an independent academic faculty position investigating host- pathogen interactions. The mentored phase of the award (K99) will be completed under the continued guidance of Dr. Herbert `Skip' Virgin in the Department of Pathology and Immunology at Washington University School of Medicine. The overall research goal of the proposal is to determine molecular mechanisms of norovirus cofactors upon viral entry. Candidate: I have a long standing interest in understanding the molecular mechanisms underlying complex host-pathogen interactions. I graduated summa cum laude from Texas A&M University with a degree in Microbiology. I subsequently joined the Molecular Microbiology Graduate Program at University of Texas Southwestern Medical School. For my doctoral thesis in Dr. Neal Alto's laboratory, I described how bacterial virulence proteins usurp the host cytoskeletal machinery and engineer pathogenic-signaling circuits within the complex environment of the cytoplasm of eukaryotic host cells. I then began a postdoctoral fellowship under the mentorship of Dr. Skip Virgin. During my fellowship in Dr. Virgin's lab, it has been my goal to couple my experiences with dissecting host-pathogen signaling networks with his ability to define the in vivo relevance of host-pathogen interactions in animal models. To this end, my research project has been focused on understanding the molecular mechanisms of murine norovirus (MNoV) replication and tropism due to its robust in vitro and in vivo systems. Specifically, we recently completed a whole-genome CRISPR screen for host genes required for MNoV replication. We discovered that MNoV binds a proteinaceous receptor, CD300lf, that is necessary both in vitro and in vivo for MNoV replication and when expressed in human cells sufficient to break the species barrier of MNoV replication. Additionally, our work described a novel, unidentified cofactor in serum required for efficient MNoV binding to cells. This work is the foundation for the research proposal outlined here. I plan to focus the remainder of my fellowship on obtaining professional skills and scientific insight necessary to transition to a tenure-track position. Career Development Plan: During my final year as a postdoctoral fellow, I will focus a significant amount of effort (15%) to developing the professional skills challenging to me that are necessary for successful independent investigators. I have assembled a career advisory committee composed of Dr. Daved Fremont, Dr. Michael Diamond, and Dr. Thaddeus Stappenbeck that will evaluate my progress in overcoming deficiencies in scientific writing and data presentation, mentoring, and laboratory management along with my scientific progress. Additionally, I will attend specific seminars both within and outside of Washington University to enhance my training and preparation for transition to independence. Lastly, I have developed a timeline with specific milestones that will guide myself, my mentor Dr. Virgin, and my career advisory committee in preparing me for my goal of successfully competing for an independent RO1 grant at the end of this four year proposal. Research Project: Murine norovirus (MNoV) is an important model for understanding human noroviruses (HNoVs) and for elucidating complex interactions between viruses, the host's microbiota, and the immune system. Recent advances in HNoV culture systems have uncovered cofactors that promote viral replication in vitro through currently unknown mechanisms. Here, we will continue our investigations into the interactions between MNoV cofactors and receptors. More specifically, the experiments outlined will directly test the novel hypothesis that norovirus tropism is determined by the combination of receptor and cofactor interactions. Our preliminary data suggests an unexplored connection between cholesterol derived bile acids, the MNoV receptor CD300lf, and host derived ceramide lipid species in promoting viral entry. Importantly, it is well established that the inability of both MNoV and HNoV to replicate in non-permissive cells are due to defects in viral entry. We will explore the interactions of each of these components in a combinatorial fashion. Furthermore, we will directly test the ability of MNoV to establish and maintain a persistent infection in mice when these pathways are perturbed using genetic and pharmacological approaches. I anticipate that our results will reveal fundamental principles of norovirus entry and provide insights into establishing a robust and reproducible in vitro HNoV replication system. More broadly speaking our hypothesis, if proven true, has the potential for establishing a novel approach for generating in vitro culture systems for currently uncultivable viruses. The initial findings of this project will help me transition to an independent academic position studying the interaction between noroviruses and their hosts. The completion of this project will not only provide novel insights into norovirus biology but the framework for a competitive RO1 application.